Synthetic oligonucleotides find widespread application in molecular biology as probes for screening complementary deoxyribonucleic acid (cDNA) and genomic DNA libraries, and as primers for DNA synthesis by DNA polymerases and reverse transcriptases. The latter applications include techniques for identifying rare messenger RNAs (mRNAs) when partial protein sequence information and a sensitive biological assay for the protein product are available, e.g. as with gamma-interferon, Gray et al., Nature, Vol.295, pgs 503-508 (1982), and techniques for sequencing DNA by the dideoxy chain termination method, e.g. Smith et al., Nucleic Acids Research, Vol. 13, pgs. 2399-2412 (1985); Schreier et al., J. Mol. Biol., Vol. 129, pgs. 169-172 (1979); and Sanger et al., J. Mol. Biol., Vol. 143, pgs. 161-178 (1980).
Recently, improvements in DNA sequencing methodologies have made use of multiple fluorescent labels to carry out sequencing automatically on a single columnar gel, e.g. Smith et al. (cited above), and Smith et al., Nature, Vol. 321, pgs. 674-679 (1986). Typically, the fluorescent labels are attached to the oligonucleotide primers used in such methods in the final step (or series of steps) in the process of constructing primers. Currently, in the phosphitetriester method, prior to removal of the freshly synthesized oligonucleotide from its support up to three additional steps are required to attach a fluorescent label: First, a linking agent is attached to the 5' end of the oligonucleotide under the same conditions used to add nucleoside phosphoramidites. Second, the linking agent is activated, e.g. by removing a protection group to expose a reactive functionality, such as a primary amine. And finally, an activated dye is reacted with the exposed functionality on the linking agent.
Additional complications can arise if one attempts to label the attached oligonucleotide with a rhodamine dye, an important class of dyes in the selection of spectrally resolvable sets of fluorescent labels, e.g. Smith et al. (cited above); and Loken et al, Cytometry, Vol. 5, pgs. 152-159 (1984). The primary reagent for cleaving an oligonucleotide from its support also chemically degrades rhodamine dyes radically altering their fluorescent properties. Thus, whenever rhodamine dyes are used in current solid phase synthesis protocols, they must be attached after the oligonucleotide has been cleaved from the solid phase support, which results in an additional step and greater inconvenience in the overall synthesis.
In view of the foregoing, the availability of dye-phosphoramidite conjugates for direct use in solid phase DNA synthesis would improve the efficiency of labeled primer synthesis by reducing the number of steps required to attach a label. In particular, some systems for DNA sequencing would be more amenable to automation if the manual liquid phase synthesis step required for rhodamine attachment could be obviated by the availability of a cleavage reagent which preserved the chemical integrity and fluorescent properties of rhodamine dyes.